Remotely programmable matrices with memories

ABSTRACT

Combinations, called matrices with memories, of matrix materials with remotely addressable or remotely programmable recording devices that contain at least one data storage unit are provided. The matrix materials are those that are used in as supports in solid phase chemical and biochemical syntheses, immunoassays and hybridization reactions. The data storage units are preferably non-volatile antifuse memories. By virtue of this combination, molecules and biological particles, such as phage and viral particles and cells, that are in proximity or in physical contact with the matrix combination can be labeled by programming the memory with identifying information and can be identified by retrieving the stored information. Combinations of matrix materials, memories, and linked molecules and biological materials are also provided. The combinations have a multiplicity of applications, including combinatorial chemistry, isolation and purification of target macromolecules, capture and detection of macromolecules for analytical purposes, selective removal of contaminants, enzymatic catalysis, chemical modification and other uses.

FIELD OF THE INVENTION

The present invention relates to the application of data storagetechnology to molecular tracking and identification. In particular,three-dimensional combinations of matrix materials with remotelyprogrammable recording devices, herein referred to as memories, areprovided. By virtue of this combination, molecules and biologicalparticles, such as phage and viral particles and cells, that are inproximity to or in physical contact with the matrix combination can beelectromagnetically tagged by programming the memory with datacorresponding to identifying information. The molecules and biologicalparticles can be identified by retrieving the stored data points andconverting them to information. Combinations of matrix materials,memories, and linked molecules and biological materials are alsoprovided. The combinations provided herein have a multiplicity ofapplications, including combinatorial chemistry, isolation andpurification of target macromolecules, capture and detection ofmacromolecules for analytical purposes, high throughput screening,selective removal of contaminants, enzymatic catalysis, chemicalmodification and other uses. These combinations are particularlyadvantageous for use in multianalyte analyses.

BACKGROUND OF THE INVENTION

There has been a convergence of progress in chemistry and biology. Amongthe important advances resulting from this convergence is thedevelopment of methods for generating molecular diversity. This advancebeen facilitated by fundamental developments in chemistry, including thedevelopment of highly sensitive analytical methods, solid state chemicalsynthesis, and sensitive and specific biological assay systems. Analysesof biological interactions and chemical reactions, however, require theuse of labels or tags to track and identify the results of suchanalyses. Typically biological reactions are monitored by radiolabels ordirect or indirect enzyme labels. Chemical reactions are also monitoredby direct or indirect means, such by linking the reactions to a secondreaction in which a colored, fluorescent, chemiluminescent or other suchproduct results. These analytical methods, however, are time consuming,tedious, and often generate high background signals. There is, thus, aneed to develop alternative methods for tracking and identifyinganalytes in biological interactions and the reactants and products ofchemical reactions.

Hybridization reactions

For example, it is often desirable to detect or quantify very smallconcentrations of nucleic acids in biological samples. Typically, toperform such measurements, the nucleic acid in the sample i.e., thetarget nucleic acid! is hybridized to a detection oligonucleotide. Inorder to obtain a detectable signal proportional to the concentration ofthe target nucleic acid, either the target nucleic acid in the sample orthe detection oligonucleotide is associated with a signal generatingreporter element, such as a radioactive atom, a chromogenic orfluorogenic molecule, or an enzyme such as alkaline phosphatase! thatcatalyzes a reaction that produces a detectable product. Numerousmethods are available for detecting and quantifying the signal.

Following hybridization of a detection oligonucleotide with a target,the resulting signal-generating hybrid molecules must be separated fromunreacted target and detection oligonucleotides. In order to do so, manyof the commonly used methods immobilize the target nucleic acids ordetection oligonucleotides on solid supports. Presently available solidsupports to which oligonucleotides are linked include nitrocellulose ornylon membranes, activated agarose supports, diazotized cellulosesupports and non-porous polystyrene latex solid microspheres. Linkage toa solid support permits fractionation and subsequent identification ofthe hybridized nucleic acids, since the target nucleic acid may bedirectly captured by oligonucleotides immobilized on solid supports.More frequently, so-called "sandwich" hybridization systems are used.These systems employ a capture oligonucleotide covalently or otherwiseattached to a solid support for capturing detectionoligonucleotide-target nucleic acid adducts formed in solution see,e.g., EP 276,302 and Gingeras et al. (1989) Proc. Natl. Acad. Sci. USA86:1173). Solid supports with linked oligonucleotides are also used inmethods of affinity purification. Following hybridization or affinitypurification, the resulting complexes or hybrids or compounds must besubjected to analyses, if identification is required.

Immunoassays

Immunoassays also detect or quantify very small concentrations ofanalytes in biological samples. Many immunoassays utilize solid supportsin which antigen, or antibody is covalently, non-covalently, orotherwise, such as via a linker, attached to a solid support. Thesupport-bound antigen or antibody is then used as an analyte in theassay. As with nucleic acid analysis, the resulting antibody-antigencomplexes or other complexes, depending upon the format used, rely onradiolabels or enzyme labels to detect such complexes.

The use of antibodies to detect and/or quantitate reagents "antigens")in blood or other body fluids has been widely practiced for many years.Two methods have been most broadly adopted. The first such procedure isthe competitive binding assay, in which conditions of limiting antibodyare established such that only a fraction usually 30-50%) of a labelede.g., radioisotope, fluorophore or enzyme) antigen can bind to theamount of antibody in the assay medium. Under those conditions, theaddition of unlabeled antigen e.g., in a serum sample to be tested) thencompetes with the labeled antigen for the limiting antibody bindingsites and reduces the amount of labeled antigen that can bind. Thedegree to which the labeled antigen is able to bind is inverselyproportional to the amount of unlabeled antigen present. By separatingthe antibody-bound from the unbound labeled antigen and then determiningthe amount of labeled reagent present, the amount of unlabeled antigenin the sample e.g., serum) can be determined.

As an alternative to the competitive binding assay, in the labeledantibody, or "immunometric" assay also known as "sandwich" assay), anantigen present in the assay fluid is specifically bound to a solidsubstrate and the amount of antigen bound is then detected by a labeledantibody see, e.g., Miles et al. (1968) Nature 29:186-189; U.S. Pat. No.3,867,517!. Using monoclonal antibodies two-site immunometric assays areavailable see, e.g., U.S. Pat. No. 4,376,110!. The "sandwich" assay hasbeen broadly adopted in clinical medicine. With increasing interest in"panels" of diagnostic tests, in which a number of different antigens ina fluid are measured, the need to carry out each immunoassay separatelybecomes a serious limitation of current quantitative assay technology.

Some semi-quantitative detection systems have been developed see, e.g.,Buechler et al. (1992) Clin. Chem. 38:1678-1684; and U.S. Pat. No.5,089,391! for use with immunoassays, but no good technologies yet existto carefully quantitate a large number of analytes simultaneously see,e.g., Ekins et al. (1990) J. Clin. Immunoassay 13:169-181! or to rapidlyand conveniently track, identify and quantitate detected analytes.

Combinatorial libraries

Drug discovery relies on the ability to identify compounds that interactwith a selected target, such as cells, an antibody, receptor, enzyme,transcription factor or the like. Traditional drug discovery involvesscreening natural products form various sources, or random screening ofarchived synthetic material. The current trend, however, is to identifysuch molecules by rational design and/or by screening combinatoriallibraries of molecules.

Methods and strategies for generating diverse libraries, primarilypeptide- and nucleotide-based oligomer libraries, have been developedusing methods of simultaneous chemical synthesis and molecular biologysee, e.g., Dower et al. (1991) Annu. Rep. Med. Chem. 26:271-280; Fodoret al. (1991) Science 251:767-773; Jung et al (1992) Angew. Chem. Ind.Ed. Engl. 31:367-383; Zuckerman et al. (192) Proc. Natl. Acad. Sci. USA89:4505-4509; Scott et al. (1990) Science 249:386-390; Devlin et al.(1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci.USA 87:6378-6382; and Gallop et al. (1994) J. Medicinal Chemistry37:1233-1251!. The resulting combinatorial libraries potentially containmillions of pharmaceutically relevant compounds and can be rapidlyscreened to identify compounds that exhibit a selected activity.

The libraries fall into roughly three categories:fusion-protein-displayed peptide libraries in which random peptides orproteins are presented on the surface of phage particles or proteinsexpressed from plasmids; support-bound synthetic chemical libraries inwhich individual compounds or mixtures of compounds are presented oninsoluble matrices, such as resin beads see, e.g., Lam et al. (1991)Nature 354:82-84! and cotton supports see, e.g., Eichler et al. (1993)Biochemistry 32:11035-11041!; and methods in which the compounds areused in solution see, e.g., Houghten et al. (1991) Nature 354:84-86,Houghten et al. (1992) BioTechniques 313:412-421; and Scott et al.(1994) Curr. Opin. Biotechnol. 5:40-48!. There are numerous examples ofsynthetic peptide and oligo-nucleotide combinatorial libraries. Thepresent direction in this area is to produce combinatorial librariesthat contain non-peptidic small organic molecules. Such libraries arebased on either a basis set of monomers that can be combined to formmixtures of diverse organic molecules or that can be combined to form alibrary based upon a selected pharmacophore monomer.

There are three critical aspects in any combinatorial library: (i) thechemical units of which the library is composed; (ii) generation andcategorization of the library, and (iii) identification of librarymembers that interact with the target of interest, and keeping track ofintermediary synthesis products and the multitude of molecules in asingle vessel.

The generation of such libraries often relies on the use of solid phasesynthesis methods, as well as solution phase methods, to producecombinatorial libraries containing tens of millions of compounds thatcan be screened in diagnostically or pharmacologically relevant in vitroassay systems. In generating large numbers of diverse molecules bystepwise synthesis, the resulting library is a complex mixture in whicha particular compound is present at very low concentrations, so that itis difficult or impossible to determine its chemical structure. Variousmethods exist for ordered synthesis by sequential addition of particularmoieties, or by identifying molecules based on spacial positioning on achip. These methods are cumbersome and ultimately impossible to apply tohighly diverse and large libraries.

Thus, an essential element of the combinatorial discovery process, aswell as other areas in which molecules are identified and tracked, isthe ability to extract the information made available during synthesisof the library or identification of the active components ofintermediary structures. While there are several techniques foridentification of intermediary products and final products,nanosequencing protocols that provide exact structures are onlyapplicable on mass to naturally occurring linear oligomers such aspeptides and amino acids. Mass spectrographic MS! analysis issufficiently sensitive to determine the exact mass and fragmentationpatterns of individual synthesis steps, but complex analytical MSstrategies are not readily automated nor conveniently performed. Also,MS analysis provides at best simple connectivity information, but nostereoisomeric information, and generally cannot discriminate amongisomeric monomers. Another problem with MS analysis is that it requirespure compounds; structural determinations on complex mixtures is eitherdifficult or impossible. Finally, MS analysis is tedious and timeconsuming. Thus, although there are a multitude of solutions to thegeneration of libraries, there are no ideal solutions to the problems ofidentification, tracking and categorization.

Similar problems arise in any screening or analytical process in whichlarge numbers of molecules or biological entities are screened. In anysystem, once a desired molecule (s) has been isolated, it must beidentified. Simple means for identification do not exist. Because of theproblems inherent in any labeling procedure, it would be desirable tohave alternative means for tracking and quantitating chemical andbiological reactions during synthesis and/or screening processes.

Therefore, it is an object herein to provide methods for identification,tracking and categorization of the components of complex mixtures ofdiverse molecules.

SUMMARY OF THE INVENTION

Combinations of (i) a miniature recording device that contains one ormore programmable data storage devices (memories) that can be remotelyprogrammed and read; and (ii) a matrix, such as a particulate supportused in chemical syntheses, are provided. The remote programming andreading is preferably effected using electromagnetic radiation.

The matrix materials matrices! are any materials that are routinely usedin chemical and biochemical synthesis. The matrix materials aretypically polymeric materials that are compatible with chemical andbiological syntheses and assays, and include, glasses, silicates,celluloses, polystyrenes, polysaccharides, sand, and synthetic resinsand polymers, including acrylamides, particularly cross-linked polymers,and other such materials. The matrices may be in the form of particlesor may be continuous in design, such as a test tube or microtiter plateor the like.

The recording device is a miniature device, typically less than 10 mm³in size, preferably smaller, that includes at least one data storageunit that includes a remotely programmable and remotely readable,preferably non-volatile, memory. This device with remotely programmablememory is in proximity with or in contact with the matrix. Inparticular, the recording device includes a memory device, preferablyhaving non-volatile memory means, for storing a plurality of data pointsand means for receiving a transmitted signal that is received by thedevice and for causing a data point corresponding to the data signal tobe permanently stored within the memory means; and, if needed, a shellthat is non-reactive with and impervious to any processing steps orsolutions in which the combination of matrix with recording device isplaced, and that is transmissive of read or write signals transmitted tothe memory. The device may also include at least one support matrixdisposed on an outer surface of the shell for retaining molecules orbiological particles.

The recording device containing the memory! is typically coated with atleast one layer of material, such as a protective polymer or a glass,including polystyrene, heavy metal-free glass, plastic, ceramic, and maybe coated with more than one layers of this and other materials. Forexample, it may be coated with a ceramic or glass, which is then coatedwith or linked to the matrix material. Alternatively, the glass orceramic or other coating may serve as the matrix.

The data storage device or memory is programmed with or encoded withinformation that identifies molecules or biological particles, either bytheir process of preparation, their identity, their batch number,category, physical or chemical properties, combinations of any of suchinformation, or other such identifying information. The molecules orbiological particles are in physical contact, direct or indirect, or inproximity with the matrix, which in turn is in physical contact or inthe proximity of the recording device that contains the data storagememory. Typically, the matrix is on the surface of the recording deviceand the molecules and biological particles are in physical contact withthe matrix material.

The matrix combinations, thus, contain a matrix material, typically inparticulate form, in physical contact with a tiny device containing oneor more remotely programmable data storage units memories!. Contact canbe effected by placing the recording device with memory on or in thematrix material or in a solution that is in contact with the matrixmaterial or by linking the device, either by direct or indirect covalentor non-covalent interactions chemical linkages or by other interactions,to the matrix.

For example, such contact is effected chemically, by chemically couplingthe device with data storage unit to the matrix, or physically bycoating the recording device with the matrix material or anothermaterial, by physically inserting or encasing the device in the matrixmaterial, by placing the device onto the matrix or by any other means bywhich the device can be placed in contact with or in proximity to thematrix material.

Thus, combinations of a miniature recording device that contains or is adata storage unit linked to or in proximity with matrices or supportsused in chemical and biotechnical applications, such as combinatorialchemistry, peptide synthesis, nucleic acid synthesis, nucleic acidamplification methods, organic template chemistry, nucleic acidsequencing, screening for drugs, particularly high throughput screening,phage display screening, cell sorting, tracking of biological particlesand other such methods, are provided. These combinations of matrixmaterial with data storage unit or recording device including the unit!are herein referred to as matrices with memories.

The matrices are either particulate of a size that is roughly 10 mm³ orsmaller or a continuous medium, such as a microtiter plate or well orplastic or other solid polymeric vial or glass vial. In instances inwhich the matrix is continuous, the data storage device memory! may beplaced in or on the matrix medium or may be embedded in the material ofthe matrix. For example, microtiter plates with the recording devicecontaining the data storage unit remotely programmable memory! embeddedin each well or vials typically with a 1 ml or smaller capacity! with anembedded recording device, may be manufactured.

The combination of matrix with memory is used by contacting it with,linking it to, or placing it in proximity with a molecule or biologicalparticle, such as a virus or phage particle, a bacterium or a cell, toproduce a second combination of a matrix with memory and a molecule orbiological particle. In certain instances, such combinations of matrixwith memory or combination of matrix with memory and molecule orbiological particle may be prepared when used or may be prepared beforeuse and packaged or stored as such for futures use.

Since matrix materials have many known uses in conjunction withmolecules and biological particles, there are a multitude of methodsknown to artisans of skill in this art for linking, joining orphysically contacting the molecule or biological particle with thematrix material. In some embodiments, the recording device with datastorage unit is placed in a solution or suspension of the molecule orbiological particle of interest. In such instances, the container, suchas the microtiter plate or test tube or other vial, is the matrixmaterial. The recording device is place in or on the matrix or can beembedded or encased in the matrix material.

The miniature recording device containing the data storage unit(s) withremotely programmable memory, includes, in addition to the remotelyprogrammable memory, means for receiving information for storage in thememory and for retrieving information stored in the memory. Such meansis typically an antenna, which also serves to provide power, that can betuned to a desired electromagnetic frequency to program the memory.Preferred frequencies are any that do not substantially alter themolecular biological interactions of interest, such as those that arenot substantially absorbed by the molecules or biological particleslinked to the matrix or in proximity of the matrix, and that do notalter the support properties of the matrix. Radio frequencies arepresently preferred, but other frequencies or optical lasers will beused, as long as the selected frequency or optical laser does notinterfere with the interactions of the molecules or biological particlesof interest. Thus, information in the form of data points correspondingto such information is stored in and retrieved from the data storagedevice by application of a selected electromagnetic radiation frequency.

The preferred miniature recording device for use in the combinationsherein is a single substrate of a size preferably less than about 10mm³, that includes a remotely programmable data storage unit(s) memory!,preferably a non-volatile memory, and an antenna for receiving ortransmitting an electromagnetic signal, preferably a radio frequencysignal; the antenna, memory and other components are preferably providedon a single substrate, thereby minimizing the size of the device. Thedevice is preferably smaller than 10 mm³ in volume, more preferably lessthan 5 mm³, most preferably about 1 mm³ or smaller, and is rapidlyprogrammable, preferably in less than 5 seconds, more preferably inabout 1 second or less. The preferred memory is non-volatile, permanent,and relies on antifuse circuitry.

Methods for electromagnetically tagging molecules or biologicalparticles are provided. Such tagging is effected by placing themolecules or biological particles of interest in proximity with therecording device or with the matrix with memory, and programming orencoding the identity of the molecule or synthetic history of themolecules or batch number or other identifying information into thememory. The, thus identified molecule or biological particle is thenused in the reaction or assay of interest and tracked by virtue of itslinkage to the matrix with memory or its proximity to the matrix withmemory, which can be queried to identify the molecule or biologicalparticle.

In particular, methods for tagging constituent members of combinatoriallibraries and other libraries or mixtures of diverse molecules andbiological particles are provided. These methods involveelectromagnetically tagging molecules, particularly constituent membersof a library, by contacting the molecules or biological particles orbringing such molecules or particles into proximity with a matrix withmemory and programming the memory with retrievable information fromwhich the identity, synthesis history, batch number or other identifyinginformation can be retrieved. The contact is preferably effected bycoating, completely or in part, the recording device with memory withthe matrix and then linking, directly or via linkers, the molecule orbiological particle of interest to the matrix support.

If the matrices are used for the synthesis of the constituent molecules,the memory of each particle is addressed and the identity of the addedcomponent is encoded in the memory at before, during, or preferablyafter! each step in the synthesis. At the end of the synthesis, thememory contains a retrievable record of all of the constituents of theresulting molecule, which can then be used, either linked to thesupport, or following cleavage from the support in an assay or forscreening or other such application. If the molecule is cleaved from thesupport with memory, the memory must remain in proximity to the moleculeor must in some manner be traceable to the molecule.

In preferred embodiments, the matrix with memory linked molecules aremixed and reacted with a sample according to a screening or assayprotocol, and those that react are isolated. The identity of reactedmolecules can then be ascertained by remotely retrieving the informationstored in the memory and decoding it to identify the linked molecules.

Compositions containing combinations of matrices with memories andcompositions of matrices with memories and molecules or biologicalparticles are also provided. In particular, coded or electronicallytagged libraries of oligonucleotides, peptides, proteins, non-peptideorganic molecules, phage display, viruses and cells are provided.Particulate matrices, such as polystyrene beads, with attached memories,and continuous matrices, such as microtiter plates or slabs, with aplurality of embedded or attached memories are provided.

These combinations of matrix materials with memories and combinations ofmatrices with memories and molecules or biological particles may be usedin any application in which support-bound molecules or biologicalparticles are used. Such applications include, but are not limited todiagnostics, such as immunoassays, drug screening assays, combinatorialchemistry protocols and other such uses. These matrices with memoriescan be used to tag cells for uses in cell sorting, to identify moleculesin combinatorial syntheses, to label monoclonal antibodies, to tagconstituent members of phage displays, in affinity separationprocedures, to label DNA and RNA, in nucleic acid amplificationreactions see, e.g., U.S. Pat. No. 5,386,024; U.S. Pat. No. U.S. Pat.No. 4,683,202 and, for example International PCT Application WO/9402634, which describes the use of solid supports in connection withnucleic acid amplification methods!, to label known compounds,particularly mixtures of known compounds in multianalyte analyses, tothereby identify unknown compounds, or to label or track unknowns andthereby identify the unknown by virtue of reaction with a known. Thus,the matrices with memories are particularly suited for high throughputscreening applications and for multianalyte analyses.

Systems and methods for recording and reading or retrieving theinformation in the data storage devices regarding the identity orsynthesis of the molecules or biological particles are also provided.The systems for recording and reading data include: a host computer orother encoder/decoder instrument having a memory for storing datarelating to the identity or synthesis of the molecules, and atransmitter means for receiving a data signal and generating a signalfor transmitting a data signal; and a recording device that includes aremotely programmable, preferably non-volatile, memory and transmittermeans for receiving a data signal and generating at least a transmittedsignal and for providing a write signal to the memory in the recordingdevice.

In particular, the systems include means for writing to and reading fromthe memory device to store and identify each of the indicators thatidentify or track the molecules and biological particles. The systemsadditionally include the matrix material in physical contact with orproximate to the recording device, and may also include a device forseparating matrix particles with memory so that each particle or can beseparately programmed.

Methods for tagging molecules and biological particles by contacting,either directly or indirectly, a molecule or biological particle with arecording device; transmitting from a host computer or decoder/encoderinstrument to the device electromagnetic radiation representative of adata signal corresponding to an indicator that either specifies one of aseries of synthetic steps or the identity or other information foridentification of the molecule or biological particle, whereby the datapoint representing the indicator is written into the memory, areprovided.

Methods for reading identifying information from recording deviceslinked to or in contact with or in proximity to a electromagneticallytagged molecule or electromagnetically tagged biological particles areprovided. These methods include the step of exposing the recordingdevice containing the memory in which the data is stored toelectromagnetic radiation EM!; and transmitting to a host computer ordecoder/encoder instrument an indicator representative of a the identityof a molecule or biological particle or identification of the moleculeor biological particle linked to or in proximity to the recordingdevice.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts combinatorial synthesis of chemical libraries on matrixsupports with memories. A, B, C . . . represent the chemical buildingblocks; a, b, c . . . represent the codes stored in memory thatcorrespond to each of A, B, C, . . . , respectively.

FIG. 2 depicts combinatorial synthesis of peptides on a matrix withmemory. Each amino acid has a corresponding code, a,b, c . . . , in thematrix memory.

FIG. 3 depicts combinatorial synthesis of oligonucleotides on matrixsupports with memories. A, G, T and C represent nucleotides, and a, g,t, and c represent the electronic codes stored in memory that correspondto each of A, G T and C,, respectively. The phosphoramidite method ofoligonucleotide synthesis is performed by methods known to those ofskill in the art see, e.g., Brown et al. (1991) "Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis" in OligonucleotidesAnalogues EDITOR: Eckstein, Fritz (Ed), IRL, Oxford, UK., pp. 1-24, esp.pp. 4-71!.

FIG. 4 depicts generation of a chemical library in which R₁, R₂, R₃, aresubstituents, each identified with a different signal, depicted as 1, 2,or 3, from the classes S₁, S₂, S₃, respectively. The circle representsan organic pharmacophore. If R₁ -R₃ are the same, and selected fromamong 50 choices, then the complete library contains 50³ =125,000members. If R₁ -R₃ different, then the resulting library hascorrespondingly more members. Each matrix memory can be encoded withinformation that represents the R_(n) added and class S_(n) ! therebyproviding a unique code for each library member.

FIG. 5 is a block diagram of the data storage means and supportingelectrical components of a preferred embodiment.

FIG. 6 is a diagrammatic view of the memory array within the recordingdevice, and the corresponding data stored in the host computer memory.

FIG. 7 is an illustration of an exemplary apparatus for separating thematrix particles with memories for individual exposure to an EM signal.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are, unless noted otherwise, incorporated byreference in their entirety.

As used herein, a matrix refers to any solid or semisolid or insolublesupport to which the memory device and/or the molecule of interest,typically a biological molecule, organic molecule or biospecific ligandis linked or contacted. Such materials include any materials that areused as affinity matrices or supports for chemical and biologicalmolecule syntheses and analyses, such as, but are not limited to:polystyrene, polycarbonate, polypropylene, nylon, glass, dextran,chitin, sand, pumice, agarose, polysaccharides, dendrimers, buckyballs,polyacrylamide, silicon, rubber, and other materials used as supportsfor solid phase syntheses, affinity separations and purifications,hybridization reactions, immunoassays and other such applications. Thematrix herein may be particulate or may be a be in the form of acontinuous surface, such as a microtiter dish or well, a glass slide, asilicon chip, a nitrocellulose sheet, nylon mesh, or other suchmaterials. When particulate, typically the particles have at least onedimension in the 5-10 mm range or smaller. Such particles, referredcollectively herein as "beads", are often, but not necessarily,spherical. Such reference, however, does not constrain the geometry ofthe matrix, which may be any shape, including random shapes, needles,fibers, elongated, etc. Roughly spherical "beads" are presentlypreferred. The "beads" may include additional components, such asmagnetic or paramagnetic particles see, e.g., Dyna beads (Dynal, Oslo,Norway)! for separation using magnets, as long as the additionalcomponents do not interfere with data entry or retrieval from thememory.

As used herein, matrix particles refer to matrix materials that are inthe form of discrete particles. The particles have any shape anddimensions, but typically have at least one dimension that is 100 mm orless, preferably 50 mm or less, more preferably 10 mm or less, andtypically have a size that is 100 mm³ or less, preferably 50 mm³ orless, more preferably 10 mm³ or less, and most preferably 1 mm³ or less.The matrices may also be continuous surfaces, such as microtiler platese.g., plates made from polystyrene or polycarbonate or deviativesthereof commerically available from Perkin Elmer Cetus, and Covalinktrays (Nunc)!, microtiler plate lids or a test tube, such as a 1 mleppendorf tube.

Matrices that are in the form of containers refers to containers, suchas test tubes and microliter plates and vials that are typically usedfor solid phase syntheses of combinatorial libraries or as vessels forscreening and diagnostic assays. Thus, a container used for chemicalsyntheses refers to a container that typically has a volume of about 1liter, generally 100 ml, and more often 10 ml or less, 5 ml or less,preferably 1 ml or less, and as small as about 50 μl-500 μl, such as 100or 250 μl. This also refers to multi-well plates, such as microtilerplates. For example, the microtiler plate will typically contain arecording device in, on, or otherwise in contact with in each of aplurality of wells.

As used herein, a matrix with a memory refers to a combination of amatrix with a miniature recording device that stores multiple bits ofdata by which the matrix may be identified, preferably in a non-volatilememory that can be written to and read from by transmission ofelectromagnetic radiation from a remote host, such as a computer. Byminiature is meant of a size less than about 10 mm³.

As used herein, a memory is a data storage unit or medium! withprogrammable memory, preferably a non-volatile memory.

As used herein, a recording device is an apparatus that includes thedata storage unit with programmable memory, and, if necessary, means forreceiving information and for transmitting information that has beenrecorded. It includes any means need for writing to and reading from thememory. The recording devices intended for use herein, are miniaturedevices that preferably are smaller than 10 mm³, and more preferably arecloser in size to 1 mm³ or smaller that contain at least one such memoryand means for receiving and transmitting data to and from the memory.

As used herein, a data storage unit with programmable memory includesany data storage means having the ability to record multiple discretebits of data, which discrete bits of data may be individually accessedread! after one or more recording operations. Thus, a matrix with memoryis a combination of a matrix material with a miniature data storageunit.

As used herein, programmable means capable of storing unique datapoints. Addressable means having unique locations that may be selectedfor storing the unique data points.

As used herein, a host computer or decoder/encoder instrument is aninstrument that has been programmed with or includes information i.e., akey! specifying the code used to encode the memory devices. Thisinstrument or one linked thereto transmits the information and signalsto the recording device and it, or another instrument, receives theinformation transmitted from the recording device upon receipt of theappropriate signal. This instrument thus creates the appropriate signalto transmit to the recording device and can interpret transmittedsignals. For example, if a "1" is stored at position 1,1 in the memoryof the recording device means, upon receipt of this information, thisinstrument or computer can determine that this means the linked moleculeis, for example, a peptide containing alanine at the N-terminus, anorganic group, organic molecule, oligonucleotide, or whatever thisinformation has been predetermined to mean. Alternatively, theinformation sent to and transmitted from the recording device can beencoded into the appropriate form by a person.

As used herein, an electromagnetic tag is a recording device that has amemory that contains unique data points that correspond to informationthat identifies molecules or biological particles linked to, directly orindirectly, in physical contact with or in proximity to the device.Thus, electromagnetic tagging is the process by which identifying ortracking information is transmitted to the recording device,

As used herein, proximity means within a very short distance, generallyless than 0.5 inch, typically less than 0.2 inches. In particular,stating that the matrix material and memory, or the biological particleor molecule and matrix with memory are in proximity means that, they areat least or at least were in the same reaction vessel or, if the memoryis removed from the reaction vessel, the identity of the vesselcontaining the molecules or biological particles with which the memorywas proximate or linked is tracked or otherwise known.

As used herein, antifuse refers to an electrical device that isinitially an open circuit that becomes a closed circuit duringprogramming, thereby providing for non-volatile memory means andpermitting remote programming and, hence identification. In practice, anantifuse is a substantially nonconductive structure that is capable ofbecoming substantially conductive upon application of a predeterminedvoltage, which exceeds a threshold voltage. It does not require anyenergy input to store the data.

As used herein, electromagnetic EM! radiation refers to radiationunderstood by skilled artisans to be EM radiation and includes, but isnot limited to radiofrequency RF!, infrared IR!, visible, ultravioletUV! radiation and laser light, coherent or otherwise.

As used herein, information identifying or tracking a biologicalparticle or molecule, refers to any information that identifies themolecule or biological particle, such as, but not limited to theidentity particle i.e. its chemical formula or name!, its sequence, itstype, its class, its purity, its properties, such as its bindingaffinity for a particular ligand. Tracking means the ability to follow amolecule or biological particle through synthesis and/or process steps.The memory devices herein store unique indicators that represent any ofthis information.

As used herein, combinatorial chemistry is a synthetic strategy thatproduces large chemical libraries. It is the systematic and repetitive,covalent connection of a set, the basis set, of different monomericbuilding blocks of varying structure to each other to produce an arrayof diverse molecules see, e.g., Gallop et al. (1994) J. MedicinalChemistry 37:1233-1251!.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid without or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, and other such biological materials.

As used herein, the molecules in the combinations include any molecule,including nucleic acids, amino acids, other biopolymers, and otherorganic molecules, including peptidomimetics and monomers or polymers ofsmall organic molecular constituents of non-peptidic libraries, that mybe identified by the methods here.

As used herein, the term "bio-oligomer" refers to a biopolymer of lessthan about 100 subunits. A bio-oligomer includes, but is not limited to,a peptide, i.e., containing amino acid subunits, an oligonucleotide,i.e., containing nucleoside subunits, a peptide-oligonucleotide chimera,and a peptidomimetic.

As used herein, the term "sequences of random monomer subunits" refersto polymers or oligomers containing sequences of monomers in which anymonomer subunit may proceed or follow any other monomer subunit.

As used herein, the term "library" refers to a collection ofsubstantially random compounds or biological particles expressing randompeptides or proteins. Of particular interest are bio-oligomers,biopolymers, or diverse organic compounds or a set of compounds preparedfrom monomers based on a selected pharmacophore.

As used herein, an analyte is any substance that is analyzed or assayedin the reaction of interest. Thus, analytes include the substrates,products and intermediates in the reaction, as well as the enzymes andcofactors.

As used herein, multianalyte analysis is the ability to measure manyanalytes in a single specimen or to perform multiple tests from a singlespecimen. The methods and combinations herein provide means to identifyor track individual analytes from among a mixture of such analytes.

As used herein, a peptidomimetic is a compound that mimics theconformation and certain stereochemical features of the biologicallyactive form of a particular peptide. In general, peptidomimetics aredesigned to mimic certain desirable properties of a compound but not theundesirable features, such as flexibility leading to a loss of thebiologically active conformation and bond breakdown. For example,methylenethio bioisostere CH₂ S! has been used as an amide replacementin enkephalin analogs see, e.g., Spatola, A. F. Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins Weinstein, B, Ed.,Vol. 7, pp. 267-357, Marcel Dekker, New York (1983); and Szelke et al.(1983) In Peptides: Structure and Function, Proceedings of the EighthAmerican Peptide Symposium, Hruby and Rich, Eds., pp. 579-582, PierceChemical Co., Rockford, Ill.!.

As used herein, complete coupling means that the coupling reaction isdriven substantially to completion despite or regardless of thedifferences in the coupling rates of individual components of thereaction, such as amino acids In addition, the amino acids, or whateveris being coupled, are coupled to substantially all available couplingsites on the solid phase support so that each solid phase support willcontain essentially only one species of peptide. Complete coupling willresult in solid phase support/first amino acid combinations.

As used herein, the biological activity or bioactivity of a particularcompound includes any activity induced, potentiated or influenced by thecompound in vivo or in vitro. It also includes the abilities, such asthe ability of certain molecules to bind to particular receptors and toinduce a functional response. It may be assessed by in vivo assays or byin vitro assays, such as those exemplified herein.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the compounds include any salts, esters or derivativesthat may be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that may beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs. For example,hydroxy groups can be esterified or etherified.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography TLC!, massspectrometry MS!, size exclusion chromatography, gel electrophoresis,particularly agarose and polyacrylamide gel electrophoresis PAGE! andhigh performance liquid chromatography HPLC!, used by those of skill inthe art to assess such purity, or sufficiently pure such that furtherpurification would not detectably alter the physical and chemicalproperties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound see, e.g., Nogrady (1985) Medicinal Chemistry ABiochemical Approach, Oxford University Press, New York, pages 388-392!.

As used herein, hydrophobic amino acids include Ala, Val, Leu, lle, Pro,Phe, Trp, and Met and any other non-naturally occurring amino acids,including as the corresponding D isomers of the hydrophobic amino acids,that have similar hydrophobic properties. It is also understood thatcertain amino acids may be replaced by substantially equivalentnon-naturally occurring variants thereof, such as D-Nva, D-Nle, D-Alle,and others listed with the abbreviations below or known to those ofskill in this art.

As used herein, the abbreviations for amino acids and protective groupsare in accord with their common usage and the IUPAC-IUB Commission onBiochemical Nomenclature see, (1972) Biochem. 11: 1726!. Each naturallyoccurring L-amino acid is identified by the standard three letter codeor the standard three letter code with or without the prefix "L-"; theprefix "D-" indicates that the stereoisomeric form of the amino acid isD. For example, as used herein, Fmoc is 9-fluorenylmethoxycarbonyl; BOPisbenzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate,DCC is dicyclohexylcarbodiimide; DDZ is dimethoxydimethylbenzyloxy; DMTis dimethoxytrityl; FMOC is fluorenylmethyloxycarbonyl; HBTU is2-(1H-benzotrialzol-1-yl)-1,1,3,3-tetramethyluronium;hexafluorophosphate NV is nitroveratryl; NVOC is6-nitroveratryloxycarbonyl and other photoremovable groups; TFA istrifluoroacetic acid; DMF for N,N-dimethylformamide; Boc istert-butoxycarbonyl; TFA for trifluoroacetic acid; HF for hydrogenfluoride; HFIP for hexafiuoroisopropanol; HPLC for high performanceliquid chromatography; FAB-MS for fast atom bombardment massspectrometry; DCM is dichloromethane, Borm is benzyloxymethyl; Pd/C ispalladium catalyst on activated charcoal; DIC isdiisopropylcarbodiimide; DCC is N,N'-dicyclohexylcarbodiimide; and For!is formyl. Protected amino acids are readily available to those of skillin this art. For example, Fmoc and Boc protected amino acids can beobtained from Fluka, Bachem, Advanced Chemtech, Sigma, CambridgeResearch Biochemical, Bachem, or Peninsula Labs or other chemicalcompanies familiar to those who practice this art.

A. Matrices

Matrices, which are generally insoluble materials used to immobilizeligands and other molecules, have application in many chemical synthesesand separations. Matrices are used in affinity chromatography, in theimmobilization of biologically active materials, and during chemicalsyntheses of biomolecules, including proteins, amino acids and otherorganic molecules and polymers. The preparation of matrices is wellknown to those of skill in this art; there are many such materials andpreparations thereof known. For example, naturally-occurring matrixmaterials, such as agarose and cellulose, may be isolated from theirrespective sources, and processed according to known protocols, andsynthetic materials may be prepared in accord with known protocols.

Matrices include any material that can act as a support matrix forattachment of the molecules or biological particles of interest and canbe in contact with or proximity to with, preferably encasing or coating,the data storage device with programmable memory. Any matrix composed ofmaterial that is compatible with and upon or in which chemical synthesesare performed, including biocompatible polymers, is suitable for useherein. These matrices, thus include any material to which the datastorage device with memory can be attached, placed in proximity thereof,impregnated, encased or otherwise connected, linked or physicallycontacted. Such materials are known to those of skill in this art, andinclude those that are used as a support matrix. These materialsinclude, but are not limited to, inorganics, natural polymers, andsynthetic polymers, including, but are not limited to: cellulose, glass,silica gels, polystyrene, polystyrene cross-linked with divinylbenzeneor the like see, Merrifield (1964) Biochemistry 3:1385-1390!,polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges,and many others.

The matrices are typically insoluble substrates that are solid, porous,deformable, or hard, and have any required structure and geometry,including, but not limited to: beads, pellets, disks, capillaries,hollow fibers, needles, solid fibers, random shapes, thin films andmembranes. Typically, when the matrix is particulate, the particles areat least about 10-2000 μM. For purposes herein, the support materialwill encase or be in contact with the data storage device, and, thus,will generally have at least one dimension on the order of 1 mm 1000 μM!or more, although smaller particles may be contacted with the datastorage devices.

Selection of these matrices will be governed, at least in part, by theirphysical and chemical properties, such as solubility, functional groups,mechanical stability, surface area swelling propensity, hydrophobic orhydrophilic properties and intended use.

The data storage device with programmable memory may be coated with amaterial, such as a glass or a plastic, that can be further derivatizedand used as the support or it may be encased, partially or completely,in the matrix material, such as during or prior to polymerization of thematerial. The data storage device with memory may be physically insertedinto the matrix material or particle. It also can be manufactured with acoating that is suitable for use as a matrix or that includes regions inthe coating that are suitable for use as a matrix. If the matrixmaterial is a porous membrane, it may be placed inside the membrane. Itis understood that when the memory device is encased in the matrix orcoated with protective material, such matrix or material must betransparent to the signal used to program the memory for writing orreading data.

In some instances, the data storage device with memory is coated with apolymer, which is then treated to contain an appropriate reactive moietyor in some cases the device may be obtained commercially alreadycontaining the reactive moiety, and may thereby serve as the matrixsupport upon which molecules or biological particles are linked.Materials containing reactive surface moieties such as amino silanelinkages, hydroxyl linkages or carboxysilane linkages may be produced bywell established surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties; N-3-(triethyoxysilyl)propyl!(phthelamic acid; andbis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing amino group reactive functionalities,include, but are not limited to, para-aminophenyltriethyoxysilane.

The data storage device with memory, however, generally should not orcannot be exposed to the reaction solution, and, thus, must be coatedwith at least a thin layer of a glass or ceramic or other protectivecoating that does not interfere with the operation of the device. Theseoperations include electrical conduction across the device andtransmission of remotely transmitted electromagnetic radiation by whichdata are written and read. It is such coating that may also serve as amatrix upon which the molecules or biological particles may be linked.

The data storage devices with memory may be coated either directly orfollowing coating with a ceramic, glass or other material, may then becoated with agarose, which is heated, the devices are dipped into theagarose, and then cooled to about room temperature. The glass or silicaor other coated semi-conductor devices, may be used as supports.

Conventional integrated circuit manufacturing and packaging methodsinclude methods and means for encapsulating integrated circuits toprotect the devices from the environment and to facilitate connection toexternal devices. Also, there are also numerous descriptions for thepreparation of semiconductor devices and wires, particularly for use asbiosensors see, e.g., U.S. Pat. No. 4,933,285; see, also Cass, Ed.(1990) Biosensors A Practical Approach, IRL Press at Oxford UniversityPress, Oxford; (biosensors are chemosensors with a biological detectionsystem, generally biologically active substances, such as enzymes,antibodies, lectins and hormone receptors, which are immobilized on thesurface of the sensor electrode or in a thin layer on the sensorelectrode)!, which measure electrochemical solution parameters, such aspH. Despite differences in the components of biosensors and recordingdevices used herein, which include memories, are remotely programmableand identify or track linked or proximate molecules and biologicalparticles and do not measure the electrochemistry of the solution, thecertain of the methods for coating electrodes and wires in the biosensorart may be adapted for use herein see, e.g., U.S. Pat. Nos. 5,342,772,5, 389,534, 5,384,028, 5,296,122, 5,334,880, 5,311,039, 4,777,019,5,143,854, 5,200,051, 5,212,050, 5,310,686, 5324,591; see, also Usmaniet al., ed. (1994) Diagnostic Biosensor Polymers, ACS Symposium SeriesNo. 556!.

It is, however, emphasized that the combinations herein are notbiosensors, which include electrodes that must be in contact with thesolution such that molecules in solution directly contact the electrode.The combinations herein are matrix materials with recording devices thatcontain data storage units that include remotely programmable memories;the recording devices used in solution must be coated with a materialthat prevents contact between the device and the solution.

1. Natural matrix support materials

Naturally-occurring supports include, but are not limited to agarose,other polysaccharides, collagen, celluloses and derivatives thereof,glass, silica, and alumina. Methods for isolation, modification andtreatment to render them suitable for use as supports is well known tothose of skill in this art see, e.g,, Hermanson et al. (1992)Immobilized Affinity Liftand Techniques, Academic Press, Inc., SanDiego!. Gels, such as agarose, can be readily adapted for use herein.Natural polymers such as polypeptides, proteins and carbohydrates;metalloids, such as silicon and germanium, that have semiconductiveproperties, as long as they do not interfere with operation of the datastorage device may also be adapted for use herein. Also, metals such asplatinum, gold, nickel, copper, zinc, tin, palladium, silver, again aslong as the combination of the data storage device with memory, matrixsupport with molecule or biological particle does not interfere withoperation of the device with memory, may be adapted for use herein.Other matrices of interest include oxides of the metal and metalloidssuch as Pt--PtO, Si--SiO, Au--AuO, TiO2, Cu--CuO, and the like. Alsocompound semiconductors, such as lithium niobate, gallium arsenide andindium-phosphide, may be used as matrices.

For example, U.S. Pat. No. 4,175,183 describes a water insolublehydroxyalkylated cross-linked regenerated cellulose and a method for itspreparation. A method of preparing the product using near stoichiometricproportions of reagents is described. Use of the product directly in gelchromatography and as an intermediate in the preparation of ionexchangers is also described.

2. Synthetic matrices

There are innumerable synthetic matrices and methods for theirpreparation known to those of skill in this art. Synthetic matrices aretypically produced by polymerization of functional matrices, orcopolymerization from two or more monomers of from a synthetic monomerand naturally occurring matrix monomer or polymer, such as agarose.Before such polymers solidify, they are contacted with the data storagedevice with memory, which can be cast into the material or dipped intothe material. Alternatively, after preparation of particles or largersynthetic matrices, the recording device containing the data storageunit(s) can be manually inserted into the matrix material. Again, suchdevices can be pre-coated with glass, ceramic, silica or other suitablematerial.

Synthetic matrices include, but are not limited to: acrylamides,dextran-derivatives and dextranco-polymers,agarose-polyacrylamideblends,other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berget al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1 st , Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) inPept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al. (1979)Isr. J. Chem. 17:243-247; Kent et al. (1978) J. Org. Chem, 43:2845-2852;Mitchell et al. (1976) Tetrahedron Lett. 42:3795-3798; U.S. Pat. No.4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat. No. 5,389,449!.Methods for preparation of such matrices are well-known to those ofskill in this art.

Synthetic matrices include those made from polymers and co-polymers suchas polyvinylalcohols, acrylates and acrylic acids such aspolyethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-etylarcrylate, polyethylene-co-methyl acrylate,polypropylene-coacrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thosecontaining acid anhydride groups such as polyethylene-co-maleicanhydride, polypropylene-co-maleic anhydride and the like. Liposomeshave also been used as solid supports for affinity purifications Powellet al. (1989) Biotechnol. Bioeng. 33:173!.

For example, U.S. Pat. No. 5,403,750, describes the preparation ofpolyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plantgrowth medium containing a hydrophilic polyurethane gel compositionprepared from chain-extended polyols; random copolymerization ispreferred with up to 50% propylene oxide units so that the prepolymerwill be a liquid at room temperature. U.S. Pat. No. 3,939,123 describeslightly crosslinked polyurethane polymers of isocyanate terminatedprepolymers comprised of poly(ethyleneoxy) glycols with up to 35% of apoly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producingthese polymers, an organic polyamine is used as a crosslinking agent.Other matrices and preparation thereof are described in U.S. Pat. Nos.4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,5,334,640, 5,328,603

U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinitychromatography, which is a polymer of an aminimide and a vinyl compoundhaving at least one pendant halo-methyl group. An amine ligand, whichaffords sites for binding in affinity chromatography is coupled to thepolymer by reaction with a portion of the pendant halo-methyl groups andthe remainder of the pendant halo-methyl groups are reacted with anamine containing a pendant hydrophilic group. A method of coating asubstrate with this polymer is also described. An exemplary aminimide is1,1 -dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compoundis a chloromethyl styrene.

U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilicpolymeric gels, preferably of a macroporous character, which carrycovalently bonded D-aminoacids or peptides which contain D-aminoacidunits. The basic carrier is prepared by copolymerization of hydroxyalkylesters or hydroxyalkylamides of acrylic and methacrylic acid withcrosslinking acrylate or methacrylate comonomers are modified by thereaction with diamines, aminoacids or dicarboxylic acids and theresulting carboxyterminal or aminoterminal groups are condensed withD-analogs of aminoacids or peptides. The peptide containing D-aminoacidsalso can be synthesized stepwise on the surface of the carrier.

U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a methodfor preparation thereof. U.S. Pat. No.4,180,524 describes chemicalsyntheses on a silica support.

3. Immobilization and activation

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports see, e.g., Mosbach(1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes,Antigens, Antibodies, and Peptides; and Kennedy et al. (1983) SolidPhase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253-391!. The most commonly used methods are absorption and adsorptionor covalent binding to the support, either directly or via a linker,such as the numerous disulfide linkages, thioether bonds, hindereddisulfide bonds, and covalent bonds between free reactive groups, suchas amine and thiol groups, known to those of skill in art see, e.g., thePIERCE CATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, whichdescribes the preparation of and use of such reagents and provides acommercial source for such reagents; and Wong (1993) Chemistry ofProtein Conjugation and Cross Linking, CRC Press!. To effectimmobilization, a solution of the protein or other biomolecule iscontacted with a support material such as alumina, carbon, anion-exchange resin, cellulose, glass or a ceramic. Fluorocarbon polymershave been used as supports to which biomolecules have been attached byadsorption see, U.S. Pat. No. 3,843,443; Published International PCTApplication WO/86 03840!.

Covalent binding of the protein or other biomolecule or organic moleculeor biological particle to chemically activated solid matrix supportssuch as glass, synthetic polymers, and cross-linked polysaccharides is amore frequently used immobilization technique. The molecule orbiological particle may be directly linked to the matrix support orlinked via linker, such as a metal see, e.g., U.S. Pat. No. 4,179,402;and Smith et al. (1992) Methods: A Companion to Methods in Enz.4:73-78!.An example of this method is the cyanogen bromide activation ofpolysaccharide supports, such as agarose. The use of perfiuorocarbonpolymer-based supports for enzyme immobilization and affinitychromatography is described in U.S. Pat. No. 4,885,250!. In this methodthe biomolecule is first modified by reaction with a perfluoroalkylatingagent such as perfluorooctylpropylisocyanate described in U.S. Pat. No.4,954,444. Then, the modified protein is adsorbed onto the fluorocarbonsupport to effect immobilization.

The activation and use of matrices are well known and may be effected byany such known methods see, e.g., Hermanson et al. (1992) ImmobilizedAffinity Ligand Techniques, Academic Press, Inc., San Diego!. Forexample, the coupling of the amino acids may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford.

B. Data storage units with memory

Any remotely programmable data storage device that can be linked to orused in proximity to the solid supports and molecules and biologicalparticles as described herein is intended for use herein. Preferreddevices are rapidly and readily programmable using penetratingelectromagnetic radiation, such as radio frequency or visible lightlasers, operate with relatively low power, have fast access 10⁻³ sec!,and are remotely programmable so that information can be stored orprogrammed and later retrieved from a distance, as permitted by the formof the electromagnetic signal used for transmission. Atransmitter/receiver system, which is preferably integrated on the samesubstrate as the memory, supplies the power to program and retrieve thedata stored in the chip memory.

This remotely programmable device can be programmed sequentially to beuniquely identifiable during and after stepwise synthesis ofmacromolecules or before or after selection of screened molecules. Incertain embodiments herein, the data storage units are informationcarriers in which the functions of writing data and reading the recordeddata are empowered by an electromagnetic signal generated and modulatedby a remote host controller. Thus, the data storage devices areinactive, except when exposed to the appropriate electromagnetic signal.In an alternative embodiment, the devices may be optically ormagnetically programmable read/write devices.

Electromagnetically programmable devices

In a preferred embodiment herein, the data storage unit includes asemiconductor chip with integrated circuits formed thereon including amemory and its supporting circuitry. These devices can be written to andinterrogated from a distance. A radiofrequeny transmitter/receiversystem supplies power to the chip to program and retrieve data. Inparticular, the data storage unit preferably includes a programmableread only non-volatile semiconductor memory PROM!, which will haveinformation describing or identifying the molecules or biologicalparticles linked to or in proximity to the matrix. This informationeither identifies the molecule or biological particles including a phageand viral particles, bacteria, cells and fragments thereof, provides ahistory of the synthesis of the molecule, or provides information, suchas a batch number or identity of the linked entity. The memory isprogrammed, before, during or, preferably, after, each step of synthesisand can thereafter be read, thereby identifying the molecule or itscomponents and order of addition, or process of synthesis.

The devices of particular interest herein rely on antifuse programmingtechnology, are about 1 mm³ in size, are rapidly programmable 1 sec orless!, can be interrogated from a distance, and are programmable usingelectromagnetic radiation, preferably of a frequency, such asradio-frequencies, that do not alter the assessed activities of themolecules and biological particles of interest. Devices that rely onother programmable memories are also intended for use herein.

Antifuses

A typical antifuse structure for use herein is formed by disposing alayer of heavily N-doped word line layer on an insulating substrate,disposing an antifuse layer of lightly N-doped programmable materialwithin an etched cavity in an overlying oxide layer, and disposing ametallic address line upon and connected to the antifuse layer. Theprogrammable material used for the antifuse layer is typically selectedfrom of silicon, germanium, carbon and alpha-tin. The properties of theprogrammable material are such that the material exhibits a relativelyhigh resistance as long as the voltage across it does not exceed athreshold level. Once the threshold voltage is exceeded, the resistanceof the material irreversibly switches form a high resistance state to arelatively low resistance state.

Antifuse structures operate by forming a conductive filament in aninitially nonconductive film. I order to increase the speed at whichantifuse circuits operate, it is desirable to reduce the resistance ofthe programmed antifuse to as low a level as possible since the higherresistance is more restrictive to electrical signals passing through itbecause higher resistance connections cause circuits to operate atslower speeds. To program or change the resistance of the antifuse froma very high level greater than 100,000,000 ohms! to a low level lessthan 1000 ohms!, a voltage of sufficiently high electrical fieldstrength is placed across the antifuse film to cause dielectricbreakdown. As breakdown occurs electrical current will flow through onesmall region of the film. The current is limited by the resistance ofthe filament itself as well as any series resistance of conductivelayers or logic devices transistors! in series with the antifuse.

Examples of the antifuse and its use as a memory cell within a Read-OnlyMemory are discussed in Roesner, "Method of Fabricating a High DensityProgrammable Read-Only Memory", U.S. Pat. No. 4,796,074 (1989) andRoesner, "Electrically Programmable Read-Only Memory Stacked above aSemiconductor Substrate", U.S. Pat. No. 4,442,507 (1984). A preferredantifuse is described in U.S. Pat. No. 5,095,362. "Method for reducingresistance for programmed antifuse" (1992). U.S. Pat. No. 5,095,362provides a method for fabricating a layer of programmable materialwithin an antifuse that exhibits relatively lower than normal resistancein its programmed state. This is accomplished by enhancing the antifusefilm with carries that become activated during the programming currentpulse. U.S. Pat. No. 5,095,362 also provides a semiconductor devicecontaining an antifuse film of the type composed of semiconductormaterial having a first electrical state that is characterized by highelectrical resistivity and a second electrical state that ischaracterized by low electrical resistivity. The material iselectrically and permanently configured to the second electrical statefrom the first electrical state upon application of a voltage exceedinga predetermined threshold voltage applied across at least a portion ofthe material. Only that portion subjected to the threshold voltage isconfigured from the first state to the second state. The remainingportion of the material, which is not exposed to the threshold voltage,remains in the first state. An element is provided for selectivelydecreasing the resistivity of the second electrical state of the portionof the material. The element for selectively decreasing resistivityincludes nonactivated conductive dopants disposed within the material.The dopants are characterized by having a nonactivated state wherein thedopants do not enhance the conduction of carriers in the film. Thedopants are further characterized by having an activated state whereinthe dopants enhance the conduction of carriers in the film. The dopantsare configured from the nonactivated state into the activated state uponapplication of a threshold voltage being applied across a predeterminedand selected portion of the material in which the dopants are disposed.The dopants are ion implanted and are selected from among boron,phosphorous and arsenic.

A preferred recording device with non-volatile, anti-fuse-based memory

Referring to FIG. 5, which depicts a preferred embodiment, a recordingdevice containing a non-volatile electrically-programmable read-onlymemory ROM! 102 that utilizes antifuse technology is combined on asingle substrate 100 with a thin-film planar antenna 110 forreceiving/transmitting an RF signal 104, a rectifier 112 for deriving avoltage from a received radio frequency RF! signal, an analog-to-digitalconverter ADC! 114 for converting the voltage into a digital signal forstorage of data in the memory, and a digital-to-analog converter DAC!116 for converting the digital data into a voltage signal fortransmission back to the host computer is provided. A single substrate100 is preferred to provide the smallest possible chip, and tofacilitate encapsulation of the chip with a protective, polymer shell orshell+matrix or matrix material! 90. Shell 90 must be non-reactive withand impervious to the various processes that the recording device isbeing used to track in order to assure the integrity of the memorydevice components on the chip. Materials for the shell include any suchmaterials that are known to those of skill in the art see, e.g., Hiroshiet al., eds. (1995) Polymneric Materials for MicroelectronicApplications: Science and Technology, ACS Symposium Ser. No. 579!,including glasses, ceramics, plastics and other inert coatings.

Based on current semiconductor integrated circuit fabrication processcapabilities, in a preferred embodiment the finished chip on which allof the listed components are integrated is on the order of 1 mm×1 mm ˜40mils×40 mils!, with a memory capacity of 1024 bits. Greater memorycapacity, where needed, and smaller chips, however, will be preferred.The chip may be larger to accommodate more memory if desired, or may besmaller as design rules permit smaller transistors and higher devicedensities.

The antifuse ROM structure described herein, and the method forfabricating the same, are based upon the teachings of U.S. Pat. No.4,424,579, issued Jan. 3, 1984, U.S. Pat. No. 4,442,507, issued Apr. 10,1984, U.S. Pat. No. 4,796,074, issued Jan. 3, 1989, and U.S. Pat. No.5,095,362, issued Mar. 10, 1992, all of Roesner, U.S. Pat. No.4,598,386, issued Jul. 1, 1986, of Roesner et al., and U.S. Pat. No.5,148,256, issued Sep. 15, 1992 and U.S. Pat. No. 5,296,722, issued Mar.22, 1994, both of Potash, et al., all of which are incorporated hereinby reference.

In an antifuse-type memory device, the individual memory cells arearranged in matrices of orthogonal conductive word and bit lines toobtain the smallest possible memory array size. For example, for 1024bits of memory, there are 32 word lines and 32 bit lines for a squarematrix. Memories with greater capacity may also be used. Schottky diodesare formed generally corresponding to the points at which the word andbit lines cross. The word and bit lines are separated by an undoped orlightly-doped semiconductor layer with interstitial doping. Each ofthese points is a memory cell and is the equivalent of a programmableswitch in series with a Schottky diode. Data are stored by the switchbeing ON or OFF. As fabricated, an antifuse memory device has all of itsswitches in the OFF state. A switch may be turned on by applying avoltage in excess of a predetermined threshold voltage to one of theword lines while setting a selected bit line to a low logic level. Thethreshold voltage is determined by the impedance of the semiconductorlayer, i.e., its doping level. According to the process for fabricatingthe antifuse memory of the preferred embodiment, the impedance can beless than 200 ohms with a threshold voltage for programming as low as 3volts. Application of voltage exceeding the threshold activates theinterstitial dopant in the semiconducting film at the pointcorresponding to the cross-over between the two lines, causing a shortbetween the word and bit lines and irreversibly turning on thatparticular switch or memory cell. Address decoders, as are known in theart, are used to selectively address the word and bit lines for purposesof both writing information to and reading stored information from thememory array. Exemplary means for decoding information to be stored inmemory and to be read from memory are provided in U.S. Pats. Nos.4,442,507 and 4,598,386.

Information to be written into the memory need not be detailed since thedata stored in the memory is primarily acting as an identificationmarker that is traceable to a more detailed record stored in the hostcomputer memory 120, independent of the memory associated with thematrix support or tagged molecule or biological particle. In thismanner, the RF signal from transmitter 80 that is used to provide thepower and the signal to the matrix particle memory need only address asingle memory cell to indicate that a nascent oligomer linked to or inproximity to the memory device has been subjected to a given processstep or to identify a molecule or biological particle. In other words, aconventional "push-pull" type of address decoder, where only one bitline and one word line are driven high and low, respectively, at anygiven time, may be used. Thus, a sophisticated memory addressing systemneed not be provided on the matrix particle memory chip, and shiftregisters may be used to control memory addressing. Alternatively, amicroprocessor which is mask-programmed during the fabrication processfor controlling an address bus which connects the ADC 114 and the DAC116 to the memory array may also be built onto the same substrate onwhich the memory and other components are integrated. Other integratedmeans for selectively addressing locations within the memory are knownand will be apparent to the practitioner skilled in the art.

Examples of alternate means for forming an antifuse memory are providedin the following U.S. Patents: U.S. Pat. No. 5,282,158, issued Jan. 25,1994, of Lee; U.S. Pat. No. 5,311,039, issued May 10, 1994, of Kimura,et al.; U.S. Pat. No. 5,334,880, issued Aug. 2, 1994, of Abadeer, et al.In addition to antifuse memory devices, other types of non-volatileelectrically-programmable read-only memories, which are known in theart, may be used. Note that non-volatility of the memory is importantsince power is applied to the chip only when it is subjected to the RFor other transmission signal.

Although antifuse memories are not designed to be erasable, it may bedesirable to re-use the devices if the memory becomes full. In suchinstances, conventional electrically programmable erasable read onlymemories EEPROMs! may be used instead. In EEPROMs, stored data can beerased by exposing the device to UV light.

Signal rectifier 112 may be a Schottky diode, making it readilyincorporated into the fabrication process used for the memory array.Other means for signal rectification may be used as are known. The ADC114 and DAC 116 are well-known devices and are readily integrated ontothe substrate 100 using the fabrication process described in thereferences for the memory array. Radio frequency modulation techniques,which are known in the art, for example, pulse code modulation, may beadapted to permit direct digital transmission, in which case the ADC andDAC may not be required.

Antenna 110 is formed during the fabrication process using conventionalphotolithographic techniques to provide one or more metal structures,such as aluminum, to receive a pre-determined wavelength RFtransmission. The antenna may be a simple straight line half-waveantenna which is created by patterning a structure during the secondmetal process steps so that the structure has a length equal to one-halfof the wavelength of the selected RF transmission frequency in freespace. Another option for formation of the antenna is as a small loop,either on a dedicated portion of the chip, or encircling the othercomponents of the chip, also formed during the second metal step of thefabrication process. It is noted that, in a typical semiconductorfabrication process, such as would be compatible with the preferredantifuse memory, the first and second metal steps include depositing alayer of aluminum, then patterning the aluminum photolithographicallyfollowed by a plasma etch to define the desired features. Except wherevias are formed, the two metal layers are separated by a dielectricfilm. Dipole antennas may be formed by patterning the second metal in asimilar manner, with the dimensions of the antenna being selected forthe appropriate RF frequency. The two metal layers may also be used toform a microstrip antenna structure by selecting the dielectric filmbetween the metal layers such that it has a dielectric constant andthickness appropriate so that the microstrip is resonant at one-half ofthe RF wavelength. The first metal layer provides the ground plane.! Themetal structures, which may be square patches, circles, lines, or othergeometries, are defined photolithographically during the normal maskingsteps of the first and second metal processes. Other antenna structureswhich can be configured as a thin film device for integration onto acommon substrate with the memory structure and other components may beused and will be apparent to those skilled in the art.

The RF antenna may, in an alternate embodiment, be formed external tothe semiconductor substrate. In this configuration, a separateconductive wire, which acts as an antenna, will be attached to a bondpad formed on the chip using methods known to those skilled in the art.The wire will then be stabilized when the chip is encased in theprotective shell, so that the antenna extends at some angle to the chip.

Also, as an alternative to signal transmission via RF, the antifuse orother semiconductor memory and supporting circuitry can receive theaddressing commands and device power by optical transmission. In thisembodiment, the RF antenna 110 would be replaced by a photocell thatgenerates sufficient write voltage to exceed the threshold voltage. Forthe addressing commands, the RF transmitter 80 is replaced by a lightsource, and the commands may be transmitted digitally by pulsing theoptical transmitter, which can be a laser, flash lamp or other highintensity light source. It is noted that the light intensity must besufficient to generate adequate voltage in the photocell to write intomemory, but not so high that it damages the metal interconnect on thechip. With digital data transmission analog-to-digital anddigital-to-analog conversion circuitry can be eliminated.

The operation of programming the memory to record the process steps towhich the linked or adjacent matrix particle or support and linked orproximate molecule or biological particle is exposed involves placingthe memory device reasonably close a distance on the order of about 1inch 25.4 mm! is presently contemplated, but longer distances should bepossible, and can be determined empirically! to RF transmitter 80. TheRF transmitter 80 emits a carrier wave modulated by a signal generatedby host computer 122 using conventional RF technology. The carrier waveitself can provide the power to the generate the programming voltage andthe operating voltage for the various devices via the rectifier, whilethe modulation signal provides the address instructions. As statedpreviously, since the memory need only be tagged to record the exposureof the proximate or linked molecule or biological particle to a givenprocess, the address signal need only carry information to turn on asingle memory location, while the host computer 122 stores into memory120 the information linking the process information with the singlememory location that was "tagged" to record exposure to the processstep. Referring to FIG. 1, in which chemical building blocks A, C, and Eare added to a molecule linked to a matrix with memory, and to FIG. 6,an illustrative example of how information is written onto a particle isprovided in Table 1.

                  TABLE 1                                                         ______________________________________                                        PROCESS                                                                       STEP    X-REGISTER ADDRESS                                                                            Y-REGISTER ADDRESS                                    ______________________________________                                        A       1               8                                                     C       2               4                                                     E       3               2                                                     ______________________________________                                    

For the step in which A is added, the address signal would increment thex-register 124 one location and increment the y-register 126 eightlocations, and then apply the programming voltage. The activation ofthis switch is indicated by an "A" at the selected address, although theactual value stored will be a binary "1", indicating ON. As described,for example, in U.S. Pat. No. 4,424,579, how the programming voltage isapplied depends on whether the decoders have depletion or enhancementtransistors.! The host computer 122 would write into its memory 120 thatfor process A, the x-,y- address is 1,8. Upon removal of the RF signalafter recording process A, the voltage is removed and the registerswould reset to 0. For the step in which C is added, the address signalwould increment the x-register 124 two locations and the y-register 126four locations, then apply the programming voltage, as indicated by theletter "C". The host computer 120 would similarly record in memory thatan indication of exposure to process C would be found at x-,y- address2,4. Again, upon removal of the RF signal, the registers reset to 0 sothat when the matrix particle's memory is again exposed to RF followingaddition of block E, the registers increment 3 and 2 locations,respectively, and the programming voltage is applied to turn on theswitch, indicated by "E". Desirably all processing steps are automated.

After processing is completed, to read the information that has beenrecorded in the memory of the data storage unit, the host computer 122will inquire into the identity of the particle by generating a commandsignal to the registers to select the appropriate address locations todetermine whether the switch is on or off. If the switch is on, i.e., avoltage drop occurs at that point, the computer will create a recordthat the particle received a particular process step. Alternatively, thehost computer can generate an inquiry signal to sequentially look at allmemory locations to determine which switches have been turned on,recording all locations at which voltage drops occurred. The computerwill then compare the "on" locations to the process steps stored in itsmemory to identify the steps through which the subject particle wasprocessed.

If desired, individual particles can be identified by reserving certainmemory locations for identification only, for example, the first tworows of the x-register. In this case, particles will be passedseparately through the RF signal while the x-register is incremented toturn on switches at address locations 0,0, 1,0, 2,0, etc. Withindividual identification, the host computer 122 can first generate asignal to query a matrix particle memory to determine its identity, thenwrite the information with regard to the process performed, saving theprocess and particle information in the host computer memory 120.

Ideally, the tagging of particles which are exposed to a particularprocess would be performed in the process vessel containing all of theparticles. The presence, however, of a large number of particles mayresult in interference or result in an inability to generate asufficiently high voltage for programming all of the particlessimultaneously. This might be remedied by providing an exposure ofprolonged duration, e.g., several minutes, while stirring the vesselcontents to provide the greatest opportunity for all particles toreceive exposure to the RF signal. On the other hand, since eachparticle will need to be read individually, a mechanism for separatingthe particles may be used in both write and read operations. Also, ininstances in which each particle will have a different moleculeattached, each particle memory must be addressed separately.

An apparatus for separating the particles to allow individual exposureto the RF signal is illustrated in FIG. 7. Here, the particles areplaced in a vessel 140 which has a funnel 142, or other constrictedsection, which permits only one particle 150 to pass at a time. It isnoted that the particles, as illustrated, are, for purposes ofexemplification, depicted as spherical. The particles, however, can beof any shape, including asymmetric shapes. Where the particles areasymmetric or of other shapes, the size of the funnel exit and tubeshould be selected to fit the largest diameter of the particles closely,If a particular orientation of the particle is desired or required foreffective transmission, the tube and funnel exit should be designed andoriented to permit only particles in the proper alignment with the tubeto exit.

The RF transmitter 80 is positioned adjacent a tube 144 which receivesinput from funnel 142. When a particle passes through tube 144 the RFtransmitter provides a signal to write to or read from the particle'smemory. Means for initiating the RF transmission may include connectionto a mechanical gate or shutter 145 in the funnel 142 which controls theadmission of the particle into the tube. As illustrated in FIG. 7,however, optical means for detecting the presence of the matrix particlewith memory to initiate RF transmission are provided in the form of alaser 146 directed toward the tube 144, which is transparent to thewavelength of the light emitted by the laser. When the laser lightimpinges upon the particle shown with dashed lines! it is reflectedtoward an optical detector 148 which provides a signal to the hostcomputer 122 to initiate the RF transmission. Alternatively, magneticmeans, or any other means for detecting the presence of the particle inthe tube 144 may be used, with the limitation that any electromagneticradiation used does not induce any reactions in the substances on theparticle's surface. After exposure of the individual particle to the RFsignal, the particle may be received in one or more vessels for furtherprocessing. As illustrated, tube 144 has an exemplary three-way splitterand selection means, shown here in dashed lines as mechanical gates, fordirecting the particles to the desired destination.

Optically or magnetically programmed devices

In addition to electrically-programmable means for storing informationon the matrix particles, optical or magnetic means may be used. Oneexample of an optical storage means is provided in U.S. Pat. No.5,136,572, issued Aug. 4, 1992, of Bradley, which is incorporated hereinby reference. Here, an array of stabilized diode lasers emits fixedwavelengths, each laser emitting light at a different wavelength. Therecording medium is photochemically active so that exposure to laserlight of the appropriate wavelength will form spectral holes. Becausedifferent wavelengths are used, multiple spectral holes can besuperimposed so that the recording medium can be very small for purposesof tagging. Further, since only a single bit of information is requiredto tag the particle at any given step, the creation of a single spectralhole at a specific wavelength is capable of providing all of theinformation needed. The host computer then makes a record associatingthe process performed with a particular laser wavelength.

For reading, the same wavelength laser that was used to create thespectral hole will be the only light transmitted through the hole. Sincethe spectral holes cannot be altered except by a laser having sufficientpower to create additional holes, this type of memory is effectivelynon-volatile. Further, the recording medium itself does not have anyoperations occurring within its structure, as is the case in electricalmemories, so its structure is quite simple. Since the recording mediumis photochemically active, it must be well encased within an opticallytransmissive, inert material to prevent reaction with the variousprocessing substances while still permitting the laser light to impingeupon the medium. Other types of optical data storage and recording mediamay be used as are known in the art. For example, optical disks, whichare typically plastic-encapsulated metals, such as aluminum, may beminiaturized, and written to and read from using conventional opticaldisk technology. In such a system, the miniature disks must be alignedin a planar fashion to permit writing and reading. A modification of thefunnel system, described above, will include a fattened tube to insurethe proper orientation. When using any form of optical recording,however, considerations must be made to insure that the selectedwavelength of light will not affect or interfere with reactions of themolecules or biological particles linked to or in proximity to matrixparticles.

C. Preparation of the combinations

1. Preparation of matrix-memory combinations

In preferred embodiments, the recording device is cast in a selectedmatrix material during manufacture. Alternatively, the devices can bephysically inserted into the matrix material, the deformable gel-likematerials, or can be placed on the matrix material and attached by aconnector, such as a plastic or wax or other such material.

2. Non-linked matrix-memory combinations

The recording device with memory can be placed onto the inner surface ofa vessel, such as a microtiter plate or vial or tube in which thereaction steps are conducted. Alternatively, the device can beincorporated into the vessel material, such into the a wall of eachmicrotiter well or vial or tube in which the reaction is conducted. Aslong as the molecules or biological particles remain associated with thewell, tube or vial, their identity can be tracked.

3. Preparation of matrix-memory-molecule or biological particlecombinations

In certain embodiments, combinations of matrices with memories andbiological particle combinations are prepared. For example, librariescan be prepared on the matrices with memories, and stored as such forfuture use or antibodies can be linked to the matrices with memories andstored for future use.

D. The System

Systems for recording and reading information are provided. The systemsinclude a host computer or decoder/encoder instrument, a transmitter, areceiver and the data storage device. The systems also can include afunnel-like device or the like for use in tagging single memory devices.In practice, an EM signal, preferably a radio frequency signal istransmitted to the data storage device. The antenna or other receivermeans in the device detects the signal and transmits it to the memory,whereby the data are written to the memory and stored in a memorylocation.

As discussed above, mixtures of the matrix with memory-linked moleculesor biological particles may be exposed to the EM signal, or each matrixwith memory (either before, after or during linkage of the biologicalparticles or molecules) may be individually exposed, using a device,such as that depicted herein, to the EM signal. Each matrix with memory,as discussed below, will be linked to a plurality of molecules orbiological particles, which may, depending upon the application beidentical or substantially identical or a mixture of molecules orbiological particles depending upon the application and protocol inwhich the matrix with memory and linked (or proximate) molecules orbiological particles is used. The memory can be programmed with dataregarding such parameters.

The location of the data, which when read and transmitted to the hostcomputer or decoder/encoderinstrument, corresponds to identifyinginformation about linked or proximate molecules or biological particles.The host computer or decoder/encoder instrument can either identify thelocation of the data for interpretation by a human or another computeror the host computer or the decoder/encoder can be programmed with a keyto interpret or decode the data and thereby identify the linked moleculeor biological particle.

E. Applications

The matrices with remotely programmable memory(ies) may be used with anymethodology that employs molecules or biological particles linked to orcombined with matrices. Examples of such methods include, but are notlimited to, methods for synthesis and screening of combinatoriallibraries and other libraries, described in the following references:U.S. Pat. Nos. 5,395,750, 5,395,587, 5,384,261, 5,359,115, 5,348,867,5,338,665, 5,316,922, 5,223,409, 5,223,408, 5,382,513, 5,260,203,5,258,289, 5,270,170, 5,288,514, 4,631,211, International Application WO94/28424, which is based on U.S. application Ser. No. 08/069,352; WO94/13623, which is based on U.S. application Ser. No. 07/988,278; andInternational Application WO 94/08051, which is based on U.S.application Ser. Nos. 08/013,948 and 07/955,371, which describessynthetic schemes for preparing libraries. Such methods also include,enzyme immunoassays, particularly ELISAs, which rely on support boundantigens, antibodies or complexes thereof; and assays and methods usinghybridizations using solid phase-bound nucleic acids. Support-boundmolecules and biological particles are often used in methods of affinitypurification. There are innumerable protocols that utilize support-boundligands of any sort, including supportbound antibodies, and supportbound nucleic acids.

Thus, there are many approaches known that rely on combinations of solidphases and molecules or biological particles, and the above listing andfollowing discussion is intended to exemplify, but not limit the methodsto which the technology provided herein is applicable.

1. Combinatorial syntheses

(a) Oligomer and polypeptide libraries

(i) Bio-oligomer libraries

One exemplary method for generating a library see, U.S. Pat. No.5,382,513! involves repeating the steps of (1) providing at least twoaliquots of a solid phase support; separately introducing a set ofsubunits to the aliquots of the solid phase support; completely couplingthe subunit to substantially all sites of the solid phase support toform a solid phase support/new subunit combination, assessing thecompleteness of coupling and if necessary, forcing the reaction tocompleteness; thoroughly mixing the aliquots of solid phase support/newsubunit combination; and, after repeating the foregoing steps thedesired number of times, removing protecting groups such that thebio-oligomer remains linked to the solid phase support. In oneembodiment, the subunit may be an amino acid, and the bio-oligomer maybe a peptide. In another embodiment, the subunit may be a nucleoside andthe bio-oligomer may be an oligonucleotide. In a further embodiment, thenucleoside is deoxyribonucleic acid; in yet another embodiment, thenucleoside is ribonucleic acid. In a further embodiment, the subunit maybe an amino acid or a nucleoside, and the bio-oligomer may be apeptide-oligonucleotide chimera. Each solid phase support is attached toa single bio-oligomer species and all possible combinations of monomersubunits of which the bio-oligomers are composed are included in thecollection.

In practicing this method herein, the support matrix has a recordingdevice with programmable memory, encased, linked or otherwise attachedto the matrix material, and at each step in the synthesis the supportmatrix to which the nascent polymer is attached is programmed to recordthe identity of the subunit that is added. At the completion ofsynthesis of each biopolymer, the resulting biopolymers linked to thesupports are mixed.

After mixing an acceptor molecule or substrate molecule of interest isadded. The acceptor molecule is one that recognizes and binds to one ormore solid phase matrix with memory/bio-oligomer species within themixture or the substrate molecule will undergo a chemical reactioncatalyzed by one or more solid phase matrix with memory/bio-oligomerspecies within the library. The resulting combinations that bind to theacceptor molecule or catalyze reaction are selected. The memory in thematrix-memory combination is read and the identity of the activebio-oligomer species is determined.

(ii) Split Bead Sequential Syntheses

Various schemes for split bead syntheses of polymers FIG. 1!, peptidesFIG. 2!, nucleic acids FIG. 3! and organic molecules based on apharmacophore monomer FIG. 4! are provided. Selected matrices withmemory particles are placed in a suitable separation, such as a funnelsee, FIG. 5!. After each synthetic step, each particle is scanned as itpasses the RF transmitter, and information identifying the addedcomponent or class of components is stored in memory. For each type ofsynthesis a code can be programmed i.e., a 1 at position 1,1 in thememory could, for example, represent alanine at the first position inthe peptide!. A host computer or decoder/encoder is programmed to sendthe appropriate signal to a transmitter that results in the appropriateinformation stored in the memory i.e., for alanine as amino acid l, a 1stored at position 1,1!. When read, the host computer or decoder/encodercan interpret the signal read from and transmitted from the memory.

In an exemplary embodiment, a selected number of beads i.e., particulatematrices with memories matrix particles linked to recording devices!,typically at least 10³ , more often 10⁴ , and desirably at least 10⁵ ormore up to and perhaps exceeding 10¹⁵, are selected or prepared. Thebeads are then divided into groups, depending upon the number of choicesfor the first component of the molecule. They are divided into a numberof containers equal to or less than for pooled screening, nestedlibraries or the other such methods! to the number of choices. Thecontainers can be microtiter wells, Merrifield synthesis vessels,columns, test tubes, gels, etc. The appropriate reagents and monomer areadded to each container and the beads in the first container are scannedwith electromagnetic with radiation, preferably high frequency radiowaves, to transmit information and encode the memory to identify thefirst monomer. The beads in the second container are so treated. Thebeads are then combined and separated according to the combinatorialprotocol, and at each stage of added monomer each separate group islabeled by inputting data specific to the monomer. At the end of thesynthesis protocol each bead has an oligomer attached and informationidentifying the oligomer stored in memory in a form that can beretrieved and decoded to reveal the identity of each oligomer.

(b) "Nested" combinatorial library protoools

In this type of protocol libraries of sublibraries are screened, and asublibrary selected for further screening see, e.g., Zuckermann et al.(1994) J. Med. Chem. 37:2678-2685; and Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646-10647!. In this method, three sets of monomers werechosen from commercially available monomers, a set of four aromatichydrophobic monomers, a set of three hydroxylic monomers, a set ofseventeen diverse monomers, and three N-termini were selected. Theselection was based on an analysis of the target receptor and knownligands. A library containing eighteen mixtures, generated from the sixpermutations of the three monomer sets, times three N-termini wasprepared. Each mixture of all combinations of the three sets of amines,four sets of hydrophobic monomers and seventeen diverse monomers wasthen assayed. The most potent mixture was selected for deconvolution bysynthesis of pools of combinatorial mixtures of the components of theselected pool. This process was repeated, until individual compoundswere selected.

Tagging the mixtures with the matrices with memories will greatlysimplify the above protocol. Instead of screening each mixtureseparately, each matrix particle with memory will be prepared with setsof the compounds, analogous to the mixtures of compounds. The resultingmatrix particles with memories and linked compounds can be combined andthen assayed. As with any of the methods provided herein, the linkedcompounds molecules or biological particles! can be cleaved from thematrix with memory prior to assaying or anytime thereafter, as long asthe cleaved molecules remain in proximity to the device or in somemanner can be identified as the molecules or particles that were linkedto the device. The matrix particle(s) with memories that exhibit thehighest affinity bind the greatest amount of sample at equilibrium! areselected and identified by querying the memory to identify the group ofcompounds. This group of compounds is then deconvoluted and furtherscreened by repeating this process, on or off the matrices withmemories, until high affinity compounds are selected.

2, Multianalyte immunoassays

The combinations of matrix with memories provided herein permits thesimultaneous assay of large numbers of analytes in a fluid. In oneexample, monoclonal antibodies very specific for carcinoembryonicantigen CEA!, prostate specific antigen PSA!, CA-125, alphafetoproteinAFP!, TGF-β, IL-2, IL-8 and IL-10 are each covalently attached to adifferent batch of matrices with memories using well-establishedprocedures and matrices for solid phase antibody assays. Eachantibody-matrix with memory complex is given a specific identificationtag, as described herein.

A sample of serum from a patient to be screened for the presence orconcentration of these antigens is added to a tube containing two ofeach antibody-matrix with memory complex a total of 16 beads, orduplicates of each kind of bead!. A mixture of monoclonal antibodies,previously conjugated to fluorescent dyes, such as fluorescein orphenyl-EDTA-Eu chelate, reactive with different epitopes on each of theantigens is then added. The tubes are then sealed and the contents aremixed for sufficient time typically one hour! to allow any antigenspresent to bind to their specific antibody-matrix with memory-antigencomplex to produce antibody-matrix with memory-antigen-labeled antibodycomplexes. At the end of the time period, these resulting complexes arebriefly rinsed and passed through an apparatus, such as that set forthin FIG. 7, but with an additional light source. As each complex passesthrough a light source, such as a laser emitting at the excitationwavelength of fluorescein, about 494 nm, or 340 nm for the Eu chelatecomplex, its fluorescence is measured and quantitated by reading theemitted photons at about 518 nm for fluorescein or 613 nm forphenyl-EDTA-Eu, and as its identity is determined by the specific signalreceived by the RF detector. In this manner, eight different antigensare simultaneously detected and quantitated in duplicate.

In an embodiment, the electromagnetically tagged matrices to recordinformation regarding linked antibodies can be used with othermultianalyte assays, such as those described by Ekins et al. (1990) J.Clin. Immunoassay 13:169-181!. These methods rely on the use of smallconcentrations of sensor-antibodies within a few μm² area. Individualmemories with matrices, or an array of memories embedded in a matrix areused. Different antibodies are linked to each memory, which isprogrammed to record the identity of the linked antibody. Alternatively,the antibody can be linked, and its identity or binding sitesidentified, and the information recorded in the memory.

In particular antibodies are linked to the matrices with memories. Thematrices are either in particular form or in the form of a slab with anarray of recording devices linked to the matrices or microtiter dish orthe like with a recording device in each well. Antibodies are thenlinked either to each matrix particle or to discrete "microspots" on theslab or in the microtiter wells. In one application, prior to use ofthese matrices with memories, they are bound to a relatively lowaffinity anti-idiotype antibody labeled with a fluorophore e.q., TexasRed, see, Ekins et al. (1990) J. Clin. Immunoassay 13:169-181!to measurethe concentration of and number of available binding sites present oneach matrix with memory particle or each microspot, which information isthen encoded into each memory for each microspot or each particle. Theselow affinity antibodies are then eluted, and the matrices can be driedand stored until used. They can be used for multianalyte analyses asdescribed above.

After reaction with the test sample, the matrices with memories arereacted with a second antibody, preferably labeled with a differentlabel, such as a different fluorophore, such as fluorescein. After thisincubation, the microspots or each matrix particle is read by passingthe particle through a laser scanner such as a confocal microscope, see,e.g., Ekins et al.(1990) J. Clin. Immunoassay 13:169-181; see also U.S.Pat. No. 5,342,633! to determine the fluorescence intensity. Thememories at each spot or linked of each particle are queried todetermine the total number of available binding sites, therebypermitting calculation of the ratio of occupied to unoccupied bindingsites.

Alternatively or additionally, the memories in the particles or at eachmicrospot could be programmed with the identity or specificity of thelinked antibody, so that after reaction with the test sample andidentification of complexed antibodies, the presence and concentrationof particular analytes in the sample can be determined.

3. Phage display

Phage, viruses, bacteria and other such manipulable hosts and vectorsreferred to as biological particles! can be modified to express selectedantigens on their surfaces by, for example, inserting DNA encoding theantigen into the host or vector genome, at a site such as in the DNAencoding the coat protein, such that upon expression the antigen ispresented on the surface of the virus, phage or bacterial host.Libraries of such packages that express diverse or families of proteinson their surfaces have been prepared. The resulting library is thenscreened with a targeted antigen and those with the highest affinity forthe antigen are selected see, e.g., U.S. Pat. Nos. 5,403,484, 5,395,750,5,382,513, 5,316,922, 5,288,622, 5,223,409, 5,223,408 and 5,348,867!.For example, libraries of synthetic antigens expressed on the surfacesof such packages have been prepared.

DNA molecules, each encoding proteins containing a family of similarpotential binding domains and a structural signal calling for thedisplay of the protein on the outer surface of a selected viral orbacterial or other package, such as a bacterial cell, bacterial spore,phage, or virus are introduced into the bacterial host, virus or phage.The protein is expressed and the potential binding domain is displayedon the outer surface of the package. The cells or viruses bearing thebinding domains to which target molecules bind are isolated andamplified, and then are characterized. One or more of these successfulbinding domains is used as a model for the design of a new family ofpotential binding domains, and the process is repeated until a novelbinding domain having a desired affinity for the target molecule isobtained.

For example, libraries of de novo synthesized synthetic antibody librarycontaining antibody fragments expressed on the surface have beenprepared. DNA encoding synthetic antibodies, which have the structure ofantibodies, specifically Fab or Fv fragments, and contain randomizedbinding sequences that may correspond in length to hypervariable regionsCDRs! can be inserted into such vectors and screened with an antigen ofchoice.

An immunoglobulin contains heavy and light chains that each containvariable and constant domains. The smallest antibody fragment that formsan antigen binding site is referred to as an Fv fragment. These Fvfragments have been prepared, using genetic engineering methods, suchthat they contain the heavy and light chain variable regions tetheredtogether by a linker, such as a flexible glycine-serine (gly-ser)_(n),in which n is 1 or more, typically 3 or 4 or more. The variable regionscan be further subdivided into framework regions that are fairly wellconserved among antibodies and hypervariable regions CDR! that are quitediverse and are important in defining antigen specificity.

Synthetic antibody libraries can be manipulated and modified for use incombinatorial type approaches in which the heavy and light chainvariable regions are shuffled and exchanged between synthetic antibodiesin order to affect specificities and affinities. This enables theproduction of antibodies that bind to a selected antigen with a selectedaffinity. The approach of constructing synthetic single chain antibodiesis directly applicable to constructing synthetic Fab fragments which canalso be easily displayed and screened. The diversity of the syntheticantibody libraries can be increased by altering the chain lengths of theCDRs and also by incorporating changes in the framework regions that mayaffect antibody affinity. In addition, alternative libraries can begenerated with varying degrees of randomness or diversity by limitingthe amount of degeneracy at certain positions within the CDRs. Thesynthetic library can be modified further by varying the chain lengthsof the CDRs and adjusting amino acids at defined positions in the CDRsor the framework region which may affect affinities. Antibodiesidentified from the synthetic antibody library can easily be manipulatedto adjust their affinity and or effector functions. In addition, thesynthetic antibody library is amenable to use in other combinatorialtype approaches. Also, nucleic acid amplification techniques have madeit possible to engineer humanized antibodies and to clone theimmunoglobulin antibody! repertoire of an immunized mouse from spleencells into phage expression vectors and identify expressed antibodyfragments specific to the antigen used for immunization see, e.g., U.S.Pat. No. 5,395,750!.

The antigens, particularly mixtures thereof, used in screening can becoupled to a solid matrix that has been combined with a recording devicecontaining an programmable PROM or EEPROM or immobilized in micotiterplates containing such devices or in a resin, such as an affigel resin,containing such device any of the preceding are matrices with memories!for use in screening antibody phage phagemids, or PAL fusion bacteria!.

Alternatively, the phage or other package can be prepared in batches andare linked to matrices that identify the DNA that has been inserted intothe phage. The matrices are then mixed and screened with labeled antigene.g., fluorescent or enzymatically! or hapten, using an assay carriedout with limiting quantities of the antigen, thereby selecting forhigher affinity phage. Thus, libraries of phage linked to matrixparticles with memories can be prepared. The matrices are encoded toidentify the batch number of the phage, a sublibrary, or to identify aunique sequence of nucleotides or amino acids in the antibody orantibody fragment expressed on its surface. The library is then screenedwith labeled antigens. The antigens are labeled with enzyme labels orradiolabels or with the antigen bound with a second binding reagent,such as a second antibody specific for a second epitope to which afluorescent antigen binds.

Following identification of antigen bound phage, the matrix particle canbe queried and the identity of the phage determined. The resultinginformation represents a profile of the sequence that binds to theantigen. This information can be analyzed using methods known to thoseof skill in this art.

4. Selection of antibodies

After cloning and plating of cells fusion cells! that secrete antibodies"panning" for the desired monoclonal or polyclonal among a vast mixtureof antibodies is a time consuming and laborious process. Attachingantibody-binding epitopes to the matrix particle with memory and"panning" with a vast number of matrix particles with memories linked toamino acids forming different epitopes or to phage particles expressingdifferent epitopes would greatly facilitate the isolation of desiredclones. After screening with the mixture of epitopes, the specificity ofthe selected antibodies could be determined by querying each memory.

Alternatively, the fused cells are plated into, for example, microtiterwells with the matrices with memory-tagged antibody binding reagent suchas protein A!. The solid phase is removed, pooled and processedbatchwise to identify the cells that produce antibodies that are thegreatest binders see, e.g., U.S. Pat. No. 5,324,633 for methods anddevice for measuring the binding affinity of a receptor to a ligand!.

5. Hybridization assays

Mixtures nucleic acid probes linked to the matrices with memories canused for screening in assays that heretofore had to be done with oneprobe at a time or with mixtures of probes followed by sequencing thehybridizing probes. There are numerous examples of such assays see,e.g., U.S. Pat. No. 5,292,874, "Nucleic acid probes to Staphylococcusaureus" to Milliman, and U.S. Pat. No. 5,232,831, "Nucleic acid probesto streptococcus pyogenes" to Milliman, et al.; see, also, U.S. Pat.Nos. 5,216,143, 5,284,747 5,352,579 and 5,374,718!. For example, U.S.Pat. No. 5,232,831 provides probes for the detection of particularStreptococcus species from among related species and methods using theprobes. These probes are based on regions of Streptococcus rRNA that arenot conserved among related Streptococcus species. Particular speciesare identified by hybridizing with mixtures of probes and ascertainingwhich probe(s) hybridize. By virtue of the instant matrices withmemories, following hybridization, the identity of the hybridizingprobes can be determined by querying the memories, and therebyidentifying the hybridizing probe.

6. Natural product screening

In the past, the vast majority of mainline pharmaceuticals have beenisolated form natural products such as plants, bacteria, fungus, andmarine microorganisms. Some of these compounds include enzymes e.g.,hyaluronidase!, industrial chemicals e.g., petroleum emulsifyingagents!, and antibiotics e.g., penicillin!. It is generally consideredthat a wealth of new agents still exist within the natural productspool. Large mixtures of natural products, even within a fermentationbroth, can be screened using the matrices with memory combinationslinked, for example, to peptides, such as antigens or antibodyfragments, of selected and known sequences. Mixtures of these peptideslinked to memory matrices can be introduced into the natural productmixture. Individual binding matrices, detected by an indicator, such asa fluorometric dye, can be isolated and the memory queried to determinewhich linked molecule or biological particle is bound to a naturalproduct.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed:
 1. A system for programming and reading data from acombination of a matrix with memory, comprising:(A) a host computer ordecoder/encoder instrument having a memory for storing data:(i) relatingto one or a plurality of process steps for synthesizing, assaying orreacting molecules or biological particles in contact with thecombination, relating to the identity of a molecule, or a biologicalparticle, or to information categorizing the molecule or biologicalparticle, wherein the biological particle is a virus, viral vector,viral capsid with or without packaged nucleic acid, phage, a phagevector, phage capsid, a cell or fragments thereof; (ii) for identifyingone or a plurality of unique indicators representative of each of theprocess steps or the identity or category of molecule or biologicalparticle; and (iii) for generating a data signal representative of eachunique indicator; (B) a transmitter means for receiving a data signaland generating a transmitted signal for transmitting a data signal, andfor providing a write signal; (C) the combination of matrix with memoryin contact with or in a solution containing a molecule or biologicalparticle, wherein the combination of matrix with memory, comprises:(i) arecording device, comprising an electromagnetically remotelyprogrammable data storage unit; and (ii) a matrix material, wherein: thematrix material is either comprised of particles of a size such that atleast one dimension is no more than 5 to 10 mm or the matrix material isin the form of a solid continuous surface that encases the recordingdevice; the matrix is derivatized for retaining products of each of thecombination of process steps, the matrix being non-reactive with theplurality of process steps; and the recording device is in contact withthe matrix or in a solution that is in contact with the matrix, andcomprises:non-volatile memory means for storing a plurality data pointsand means for receiving the transmitted signal so that the write signalcauses a stored data point corresponding to the data signal to be storedwithin the non-volatile memory means; (D) means for reading from thenon-volatile memory means to identify each of the stored data points,wherein the stored data points indicate exposure of the combination ofmatrix with memory to one of a combination of process steps or theidentity or category of the molecule or biological particle.
 2. Thesystem of claim 1, wherein the non-volatile memory means comprises anelectrically-programmable read-only memory having a plurality of uniqueaddresses.
 3. The system of claim 1, wherein the non-volatile memorymeans comprises an optically-programmable memory having a plurality ofunique addresses.
 4. The system of claim 2, wherein each data signalincludes address data corresponding to one of the plurality of uniqueaddresses.
 5. A system as in claim 2, wherein the remotelyelectrically-remotely programmable memory comprises a plurality ofantifuses and the write signal comprises a voltage greater than athreshold voltage for each of the plurality of antifuses.
 6. A system asin claim 2, wherein the transmitter means comprises a radio frequencytransmitter and the means for receiving the transmitted signal comprisesa radio frequency antenna.
 7. The system of claim 3, wherein thetransmitter means comprises a modulated light source and the means forreceiving the transmitted signal comprise a photocell.
 8. The system ofclaim 1, wherein, the host computer or decoder/encoder instrument andthe transmitter means are the means for reading from the non-volatilememory means, wherein the host computer or decoder/encoder instrumentfurther generates an inquiry signal for determining the presence orabsence of each data point within the non-volatile memory means and thetransmitter means further generates a transmitted inquiry signal fortransmitting the inquiry signal to the non-volatile memory means andreceives a response to the transmitted inquiry signal indicative of thepresence or absence of each data point.
 9. The system of claim 8,wherein the inquiry signal includes an address signal within thenon-volatile memory means corresponding to a location of each datapoint.
 10. The system of claim 1, further comprising an apparatus forseparating a single recording device or matrix with memory from aplurality of recording devices or matrices with memories to facilitatewriting to and reading from the single recording device or matrix withmemory.
 11. The system of claim 10, wherein the apparatus forseparating, comprises:a funnel means attached to a vessel for receivingthe single memory device, the funnel means having an exit in theconstriction opening of a diameter sufficient to permit only onerecording device to pass through the opening; a tube connected to afirst end of the exit opening of the funnel means for receiving thesingle recording device or matrix with memory, the tube having adiameter substantially equal to the constriction diameter; a means fordetermining a presence of the single recording device or matrix withmemory in the tube and for providing an activation signal forcontrolling the transmitter means; and at least one receiving vesselconnected at a second end of the tube for receiving the single memorydevice or matrix with memory.
 12. The system of claim 11, wherein theshapes of exit opening and the tube match a cross-sectional shape of thesingle recording device.
 13. The method of claim 1, wherein the deviceis less than about 10 mm³ in size.